Characterization of the Pharmacological-Sensitivity Profile of Neoglycoprotein-Induced Acrosome Reaction in Mouse Spermatozoa1

Abstract

Mammalian spermatozoa undergo the acrosome reaction (AR) in response to the interaction of a carbohydrate-recognizing molecule(s) on the sperm plasma membrane (sperm surface receptor) and its complementary glycan (ligand) moiety(ies) on the zona pellucida (ZP). Previously, we demonstrated that a hexose (mannose) or two amino sugars (glucosaminyl or galactosaminyl residues) when covalently conjugated to a protein backbone (neoglycoproteins) mimicked the mouse ZP3 glycoprotein and induced the AR in capacitated mouse spermatozoa (Loeser and Tulsiani, Biol Reprod 1999; 60:94-101). To elucidate the mechanism underlying sperm-neoglycoprotein interaction and the induction of the AR, we have examined the effect of several AR blockers on neoglycoprotein-induced AR. Our data demonstrate that two known L-type Ca(2+) channel blockers prevented the induction of the AR by three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA). The fact that the L-type Ca(2+) channel blockers (verapamil, diltiazem) had no inhibitory effect on sperm surface galactosyltransferase or alpha-D-mannosidase, two carbohydrate-recognizing enzymes thought to be sperm surface receptors, suggests that the reagents block the AR by a mechanism other than binding to the active site of the enzymes.

The Contribution of d-Mannose, l-Fucose, N-Acetylglucosamine, and Selectin Residues on the Binding of Glycodelin Isoforms to Human Spermatozoa1

“…the action of ZP3 glycoprotein and induce acrosome reaction in capacitated mouse [28,40] and human sperm [50]. The acrosome reaction-inducing ability of neoglycoprotein and the reduction of glycodelin binding after acrosome reaction requires the use of fixed, instead of live, sperm for characterization of the glycodelin-binding sites on sperm using neoglycoproteins; otherwise, equilibrium binding would have been impossible to achieve.…”

“…Neoglycoproteins are synthesized by linkage of specific carbohydrate residues to a protein backbone. They have been used to investigate the impact of a given carbohydrate moiety in cell-to-cell interactions [40,41]. The third objective of this study was to address the role of a single carbohydrate in neoglycoproteins and of selectins in the binding of glycodelin-A and -F to sperm.…”

Identification and Localization of T-type Voltage-operated Calcium Channel Subunits in Human Male Germ Cells

“…NGPs, which have been proposed to act in a similar manner to human ZP, induce elevation of [Ca 2ϩ ] i and AR in human spermatozoa (37,43,44). NGP-induced AR is blocked by VOCC antagonists (44,45) and mibefradil, a semispecific blocker of T-type channels, blocks NGP-induced [Ca 2ϩ ] i signaling and AR with similar potency (37,46). Using molecular and immunohistochemical techniques, Benoff and colleagues (27) have provided evidence for the presence of a number of isoforms of the HVA ␣ 1C subunit in human testis and spermatozoa.…”

Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida

“…In result, sperm -ZP binding was significantly decreased in the presence of BSA-Fuc (2.5-250 lg ml À1 ) or BSA-Gal (25-250 lg ml À1 ) (Fig. Although several kinds of monosugar linked to a BSA and or Lewis X -BSA mimicked ZP3 in mouse (Loeser et al, 1999;Hanna et al, 2004), we firstly reported that fucose-BSA can also inhibit sperm-ZP binding in mouse. Because the assay medium contained an excess amount of BSA, binding inhibition by these NGPs was not due to free BSA.…”

“…Glycans with a terminal Lewis X (Galb4[Fuca3]GlcNAc-R) or Lewis A (Galb3[Fuca4]GlcNAc) structure act as specific and potent inhibitors of a subset of sperm binding to ZP3 in mice (Johnston et al, 1998). Several studies have investigated whether neoglycoproteins (NGPs) synthesised by the linkage of specific carbohydrate residues to a protein backbone can mimic natural ZP glycoconjugates (Benoff et al, 1993(Benoff et al, , 1997Brandelli et al, 1995;Blackmore & Eisoldt, 1999;Loeser et al, 1999;Hanna et al, 2004;Rosano et al, 2007). Removal of the fucosyl residue from the Lewis X trisaccharide abolishes the glycan's ability to act as a competitive inhibitor of ZP3 binding.…”